RESUMEN
BACKGROUND: Outbred mice exhibit increased airway and intestinal immunoglobulin A (IgA) following injury when fed normal chow, consistent with humans. Parenteral nutrition (PN) eliminates IgA increases at both sites. Inbred mice are needed for detailed immunological studies; however, specific strains have not been evaluated for this purpose. BALB/c and C57BL/6 are common inbred mouse strains but demonstrate divergent immune responses to analogous stress. This study addressed which inbred mouse strain best replicates the outbred mouse and human immune response to injury. METHODS: Intravenously cannulated mice received chow or PN for 5 days and then underwent sacrifice at 0 or 8 hours following controlled surgical injury (BALB/c: n = 16-21/group; C57BL/6: n = 12-15/group). Bronchoalveolar lavage (BAL) was analyzed by enzyme-linked immunosorbent assay for IgA, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6, while small intestinal wash fluid (SIWF) was analyzed for IgA. RESULTS: No significant increase in BAL IgA occurred following injury in chow- or PN-fed BALB/c mice (chow: P = .1; PN: P = .7) despite significant increases in BAL TNF-α and SIWF IgA (chow: 264 ± 28 vs 548 ± 37, P < .0001; PN: 150 ± 12 vs 301 ± 17, P < .0001). Injury significantly increased mucosal IgA in chow-fed C57BL/6 mice (BAL: 149 ± 33 vs 342 ± 87, P = .01; SIWF: 236 ± 28 vs 335 ± 32, P = .006) and BAL cytokines. After injury, PN-fed C57BL/6 mice exhibited no difference in BAL IgA (P = .9), BAL cytokines, or SIWF IgA (P = .1). CONCLUSIONS: C57BL/6 mice exhibit similar airway responses to injury as outbred mice and humans, providing an appropriate model for studying mucosal responses to injury. The BALB/c mucosal immune system responds differently to injury and does not replicate the human injury response.
Asunto(s)
Nutrición Enteral/métodos , Inmunidad Innata , Inmunidad Mucosa , Nutrición Parenteral/métodos , Herida Quirúrgica/inmunología , Animales , Lavado Broncoalveolar , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina A/inmunología , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Intestino Delgado/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVE: To define gut-associated lymphoid tissue (GALT) phenotype changes with parenteral nutrition (PN) and PN with bombesin (BBS). BACKGROUND: PN reduces respiratory tract (RT) and GALT Peyer patch and lamina propria lymphocytes, lowers gut and RT immunoglobulin A (IgA) levels, and destroys established RT antiviral and antibacterial immunity. BBS, an enteric nervous system neuropeptide, reverses PN-induced IgA and RT immune defects. METHODS: Experiment 1: Intravenously cannulated ICR mice received chow, PN, or PN + BBS injections for 5 days. LSR-II flow cytometer analyzed Peyer patches and lamina propria isolated lymphocytes for homing phenotypes (L-selectin and LPAM-1) and state of activation (CD25, CD44) in T (CD3)-cell subsets (CD4 and CD8) along with homing phenotype (L-selectin and LPAM-1) in naive B (IgD) and antigen-activated (IgD or IgM) B (CD45R/B220) cells. Experiment 2: Following the initial experiment 1 protocol, lamina propria T regulatory cell phenotype was evaluated by Foxp3 expression. RESULTS: Experiment 1: PN significantly reduced lamina propria (1) CD4CD25 (activated) and (2) CD4CD25LPAM-1 (activated cells homed to the lamina propria) T cells, whereas PN-BBS assimilated chow levels. PN significantly reduced lamina propria (1) IgD (naive), (2) IgDLPAM (antigen-activated homed to the lamina propria) and CD44 memory B cells, whereas PN-BBS assimilated chow levels. Experiment 2: PN significantly reduced lamina propria CD4CD25Foxp3 T regulatory cells compared with chow-fed mice, whereas PN + BBS assimilated chow levels. CONCLUSIONS: PN reduces lamina propria activated and T regulatory cells and also naive and memory B cells. BBS addition to PN maintains these cell phenotypes, demonstrating the intimate involvement of the enteric nervous system in mucosal immunity.
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Bombesina/administración & dosificación , Inmunidad Mucosa/inmunología , Mucosa Intestinal/inmunología , Subgrupos Linfocitarios/inmunología , Neuropéptidos/administración & dosificación , Nutrición Parenteral Total , Mucosa Respiratoria/inmunología , Animales , Inmunidad Mucosa/efectos de los fármacos , Inmunoglobulina A/inmunología , Mucosa Intestinal/efectos de los fármacos , Subgrupos Linfocitarios/efectos de los fármacos , Masculino , Ratones Endogámicos ICR , Modelos Animales , Fenotipo , Mucosa Respiratoria/efectos de los fármacosRESUMEN
BACKGROUND: Parenteral nutrition (PN) increases infectious risk in critically ill patients compared with enteral feeding. Previously, we demonstrated that PN feeding suppresses the concentration of the Paneth cell antimicrobial protein secretory phospholipase A2 (sPLA2) in the gut lumen. sPLA2 and other Paneth cell proteins are released in response to bacterial components, such as lipopolysaccharide (LPS), and they modulate the intestinal microbiome. Because the Paneth cell protein sPLA2 was suppressed with PN feeding, we hypothesized PN would diminish the responsiveness of the small bowel to LPS through reduced secretions and as a result exhibit less bactericidal activity. METHODS: The distal ileum was harvested from Institute of Cancer Research mice, washed, and randomized for incubation with LPS (0, 1, or 10 µg/mL). Culture supernatant was collected and sPLA2 activity was measured. Bactericidal activity of the ileum segment secretions was assessed against Pseudomonas aeruginosa with and without an sPLA2 inhibitor at 2 concentrations, 100 nmol/L and 1 µmol/L. Institute of Cancer Research mice were randomized to chow or PN for 5 days. Tissue was collected for immunohistochemistry (IHC) and ileal segments were incubated with LPS (0 or 10 µg/mL). sPLA2 activity and bactericidal activity were measured in secretions from ileal segments. RESULTS: Ileal segments responded to 10 µg/mL LPS with significantly greater sPLA2 activity and bactericidal activity. The bactericidal activity of secretions from LPS stimulated tissue was suppressed 50% and 70%, respectively, with the addition of the sPLA2-inhibitor. Chow displayed greater sPLA2 in the Paneth cell granules and secreted higher levels of sPLA2 than PN before and after LPS. Accordingly, media collected from chow was more bactericidal than PN. IHC confirmed a reduction in Paneth cell granules after PN. CONCLUSION: This work demonstrates that ileal segments secrete bactericidal secretions after LPS exposure and the inhibition of the Paneth cell antimicrobial protein sPLA2 significantly diminishes this. PN feeding resulted in suppressed secretion of the sPLA2 and resulted in increased bacterial survival. This demonstrates that PN significantly impairs the innate immune response by suppressing Paneth cell function.
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Fosfolipasas A2 Grupo II/metabolismo , Íleon/inmunología , Células de Paneth/metabolismo , Nutrición Parenteral/efectos adversos , Pseudomonas aeruginosa/crecimiento & desarrollo , Alimentación Animal , Animales , Biomarcadores/metabolismo , Western Blotting , Recuento de Colonia Microbiana , Fosfolipasas A2 Grupo II/antagonistas & inhibidores , Íleon/metabolismo , Íleon/microbiología , Lipopolisacáridos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos ICR , Distribución Aleatoria , Salmonella entericaRESUMEN
BACKGROUND: Ventilator-associated pneumonia (VAP) complicates the clinical course of critically injured intubated patients. Bronchoscopic bronchoalveolar lavage (BAL) represents an invasive and accurate means of VAP diagnosis. Unilateral and blinded techniques offer less invasive alternatives to bronchoscopic BAL. This study evaluated clinical criteria as well as unilateral directed versus bilateral BAL for VAP diagnosis. METHODS: A retrospective chart review of 113 consecutive intubated trauma patients with clinically suspected VAP undergoing unilateral versus bilateral BAL was performed with comparison of positive culture results (>10(4) colony-forming units [CFU]/mL). Culture results were compared with chest radiograph (CXR) infiltrates and white blood cell (WBC) count elevation. RESULTS: Bilateral BAL was more likely to be positive than unilateral BAL (50.4% vs. 25.5%). In 37.1% of bilateral BALs, there was discordance between the sides of positivity or the bacteria isolated. A CXR infiltrate and WBC count elevation did not predict positive BAL. CONCLUSIONS: Clinical indicators of VAP are inaccurate, and bilateral bronchoscopic BAL is more likely than unilateral BAL to provide a positive sample in intubated trauma patients. Techniques that do not sample both lungs reliably should be avoided for diagnosis in this patient population.
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Lavado Broncoalveolar/métodos , Neumonía Asociada al Ventilador/diagnóstico , Adulto , Bacterias/clasificación , Bacterias/aislamiento & purificación , Líquido del Lavado Bronquioalveolar/microbiología , Distribución de Chi-Cuadrado , Recuento de Colonia Microbiana , Femenino , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Neumonía Asociada al Ventilador/sangre , Neumonía Asociada al Ventilador/microbiología , Radiografía Torácica , Estudios Retrospectivos , Ventiladores MecánicosRESUMEN
BACKGROUND: Both humans and mice increase airway immunoglobulin A (IgA) after injury. This protective response is associated with TNF-α, IL-1ß, and IL-6 airway increases and in mice is dependent upon these cytokines as well as enteral feeding. Parenteral nutrition (PN) with decreased enteral stimulation (DES) alters gut barrier function, decreases intestinal IgA, and decreases the principal IgA transport protein pIgR. We investigated the small intestine (SI) IgA response to injury and the role of TNF-α, IL-1ß, IL-6, and PN/DES. METHODS: Expt 1: Murine kinetics of SI washing fluid (SIWF) IgA; SI, SIWF and serum TNF-α, IL-1ß, and IL-6, was determined by ELISA from 0 to 8 hours after a limited surgical stress injury (laparotomy and neck incisions). Expt 2: Mice received chow or PN/DES before injury and SIWF IgA and SI pIgR levels were determined at 0 and 8 hours. Expt 3: Mice received PBS, TNF-α antibody, or IL-1ß antibody 30 minutes before injury to measure effects on the SIWF IgA response. Expt 4: Mice received injury or exogenous TNF-α, IL-1ß, and IL-6 to measure effects on the SIWF IgA response. RESULTS: Expt 1: SIWF IgA levels increased significantly by 2 hours after injury without associated increases in TNF-α or IL-1ß whereas IL-6 was only increased at 1 hour after injury. Expt 2: PN/DES significantly reduced baseline SIWF IgA and SI pIgR and eliminated their increase after injury seen in Chow mice. Expt 3: TNF-α and IL-1ß blockade did not affect the SIWF IgA increase after injury. Expt 4: Exogenous TNF-α, IL-1ß, and IL-6 increased SIWF IgA similarly to injury. CONCLUSION: The SI mucosal immune responds to injury or exogenous TNF-α, IL-1ß, and IL-6 with an increase in lumen IgA, although it does not rely on local SI increases in TNF-α or IL-1ß as it does in the lung. Similar to the lung, the IgA response is eliminated with PN/DES.
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Inmunoglobulina A/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/lesiones , Intestino Delgado/inmunología , Intestino Delgado/lesiones , Nutrición Parenteral , Animales , Anticuerpos/farmacología , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/fisiología , Interleucina-1beta/sangre , Interleucina-1beta/inmunología , Interleucina-1beta/farmacología , Interleucina-6/sangre , Interleucina-6/farmacología , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos , Modelos Animales , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: The function of secretory phospholipase A2 (sPLA2) is site dependent. In tissue, sPLA2 regulates eicosanoid production; in circulation, sPLA2 primes neutrophils; and in the intestinal lumen, sPLA2 provides innate bactericidal immunity as a defensin-related protein. Since parenteral nutrition (PN) primes leukocytes while suppressing intraluminal mucosal immunity, the authors hypothesized that (1) PN would diminish luminal sPLA2 activity but increase activity in intestinal tissue and serum and (2) stress would accentuate these changes. METHODS: Mice received chow, a complex enteral diet (CED), intragastric PN (IG-PN), or PN in experiment 1 and chow, chow+stress, PN, or PN+stress in experiment 2. RESULTS: In experiment 1, luminal sPLA2 activity was greatest in chow and decreased in CED, IG-PN, and PN, with PN lower than CED and IG-PN. Compared to that after chow, serum sPLA2 activity dropped after CED, IG-PN, and PN. Serum sPLA2 was higher in portal than systemic serum. In experiment 2, PN lowered luminal sPLA2 activity vs chow. Stress lowered luminal sPLA2 activity in chow, without change in PN. Following stress, luminal immunoglobulin A increased in chow but not PN. Serum sPLA2 activity increased in PN. CONCLUSIONS: PN attenuates sPLA2 activity in intestinal fluid, consistent with suppressed innate mucosal defense. Stress suppresses luminal fluid sPLA2 activity in chow but not the immunoglobulin A response; PN impairs both. Stress significantly elevates serum sPLA2 in PN-fed mice, consistent with known increased neutrophil priming with PN. PN reduces innate bactericidal immunity of the gut but upregulates serum proinflammatory products poststress.
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Inmunidad Innata/fisiología , Mucosa Intestinal/enzimología , Intestino Delgado/enzimología , Nutrición Parenteral , Fosfolipasas A2 Secretoras/metabolismo , Sistema Porta/inmunología , Estrés Fisiológico/inmunología , Animales , Bacterias , Nutrición Enteral , Inmunidad Mucosa , Inmunoglobulina A/metabolismo , Mediadores de Inflamación/sangre , Mucosa Intestinal/inmunología , Intestino Delgado/inmunología , Ratones , Nutrición Parenteral/efectos adversos , Fosfolipasas A2 Secretoras/sangre , Fosfolipasas A2 Secretoras/inmunología , Sistema Porta/metabolismoRESUMEN
OBJECTIVE: To determine effects of (1) parenteral nutrition (PN), (2) exogenous Lymphotoxin ß receptor (LTßR) stimulation in PN animals, and (3) exogenous LTßR blockade in chow animals on NF-κB activation pathways and products: MAdCAM-1, chemokine (C-C motif) Ligand (CCL) 19, CCL20, CCL25, interleukin (IL)-4, and IL-10. BACKGROUND: LT stimulates LTßR in Peyer's patches (PP) to activate NF-κB via the noncanonical pathway. The p100/RelB precursor yields p52/RelB producing MAdCAM-1, cytokines, and chemokines important in cell trafficking. TNFα, IL-1ß, and bacterial products stimulate the inflammatory canonical NF-κB pathway producing p65/p50 and c-Rel/p50. PN decreases LTßR, MAdCAM-1, and chemokines in PP and lowers small intestinal IgA compared with chow. METHODS: Canonical (p50 and p65) and noncanonical (p52 and Rel B) NF-κB proteins in PP were analyzed by TransAM NF-κB kit after 5 days of chow or PN, 2 days of LTßR stimulation or 3 days of LTßR blockade. MAdCAM-1, chemokines, and cytokines in PP were measured by ELISA after LTßR stimulation or blockade. RESULTS: PN significantly reduced all NF-κB proteins in PP compared with chow. Exogenous LTßR stimulation during PN increased p50, p52, Rel B, MAdCAM-1, IL-4, and IL-10 in PP, but not p65, CCL19, CCL20, or CCL25 compared with PN. LTßR blockade reduced noncanonical products (p52 and Rel B), MAdCAM-1, CCL19, CCL20, CCL25, IL-4, and IL-10 but had no effect on the inflammatory pathway (p50 and p65) compared with chow. CONCLUSION: Lack of enteral stimulation during PN decreases both canonical and noncanonical NF-κB pathways in PP. LTßR stimulation during PN feeding completely restores PP noncanonical NF-κB activity, MAdCAM-1, IL-4, IL-10, and partly the canonical pathway. LTßR blockade decreases the noncanonical NF-κB activity, MAdCAM-1, chemokines, and cytokines without effect on the canonical NF-κB activity in PP.
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Receptor beta de Linfotoxina/metabolismo , FN-kappa B/metabolismo , Nutrición Parenteral/efectos adversos , Transducción de Señal , Análisis de Varianza , Animales , Moléculas de Adhesión Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inmunoglobulinas/metabolismo , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Mucosa Intestinal/metabolismo , Receptor beta de Linfotoxina/fisiología , Masculino , Ratones , Ratones Endogámicos , Mucoproteínas/metabolismo , Nutrición Parenteral/métodos , Distribución Aleatoria , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Secretory immunoglobulin A (sIgA) increases in the airways of humans and mice after injury to protect against infection. The pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 are linked molecularly to sIgA production and secretion and are required for sIgA increases in the airway after injury in a mouse model. We investigated the injury effect on airway and serum concentrations to determine the source of the cytokines involved in the airway IgA response. METHODS: In the first experiment, TNF-α, IL-1ß, and IL-6 concentrations in bronchoalveolar lavage (BAL) fluid and serum obtained from 11 ventilated trauma patients within 30 h of admission were compared with those in eight elective surgical patients. In the second experiment, male ICR mice received no injury (n = 7) or injury with sham celiotomy and neck incisions (n = 8) with sacrifice of all animals at 8 h for BAL fluid and serum cytokine measurements by enzyme-linked immunosorbent assay. RESULTS: Injured patients had significantly higher BAL fluid and serum TNF-α, IL-1ß, and IL-6 concentrations, with greater increases in the BAL fluid than in the serum. Injured mice had significantly increased BAL fluid concentrations of TNF-α, IL-1ß, and IL-6 without significant changes in serum TNF-α or IL-1ß. Serum IL-6 increased significantly. CONCLUSIONS: Injury significantly increases human and mouse airway TNF-α, IL-1ß, and IL-6. Increases are greater in the airway than in serum, implying a local rather than a systemic stress response to injury.
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Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Sistema Respiratorio/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Heridas y Lesiones/inmunología , Adolescente , Adulto , Anciano de 80 o más Años , Animales , Sangre/inmunología , Análisis Químico de la Sangre , Líquido del Lavado Bronquioalveolar/química , Femenino , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
BACKGROUND: A common problem that complicates use of central venous access devices (CVADs) is occlusion by thrombosis. Alteplase, a recombinant tissue plasminogen activator, is used to restore line patency when thrombosis occurs. Heparin flush is commonly used to prevent this complication, but the effectiveness of this practice is unclear. A recent heparin shortage allowed examination of heparin effectiveness in reducing CVAD thrombosis. METHODS: A retrospective cohort study was performed by querying a pharmacy database for alteplase use for CVAD thrombosis in adult patients during periods when heparin flushes (10 units/mL) were used and when saline flushes were used instead because of a nationwide heparin shortage. The number of patients receiving alteplase, the number of doses administered, and the total amount of alteplase used were compared over 1-month intervals of heparin flush use and 1-month intervals of saline flush use. Patient days and critical care patient days were compared between these time intervals. Peripherally inserted central catheter (PICC) line placements and replacements between time periods of heparin and saline flush were also compared. RESULTS: Significant increases in the number of patients receiving alteplase (P = .04), the number of alteplase doses administered (P = .04), and total dose of alteplase used (P = .05) occurred during the heparin shortage. No significant differences in patient population were observed. The percentage of PICC line replacements also increased significantly (P < .05) when heparin was not available. CONCLUSIONS: Heparin flush (10 units/mL) decreases thrombotic occlusions of CVADs, resulting in decreased alteplase use and fewer PICC line replacements.
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Cateterismo Venoso Central/efectos adversos , Catéteres de Permanencia/efectos adversos , Fibrinolíticos/administración & dosificación , Heparina/administración & dosificación , Trombosis/prevención & control , Activador de Tejido Plasminógeno/administración & dosificación , Grado de Desobstrucción Vascular/efectos de los fármacos , Adulto , Cateterismo Venoso Central/métodos , Humanos , Tiempo de Internación , Estudios Retrospectivos , Trombosis/tratamiento farmacológico , Resultado del TratamientoRESUMEN
BACKGROUND: : Injury stimulates an innate airway IgA response in severely injured patients, which also occurs in mice. Tumor necrosis factor (TNF)-α and interleukin (IL)-1ß stimulate the production of polymeric immunoglobulin receptor, the protein required to transport immunoglobulin A (IgA) to mucosal surfaces. Blockade of TNF-α and IL-1ß eliminates the airway IgA response to injury. IL-6 stimulates differentiation of B cells into IgA-secreting plasma cells at mucosal sites. We investigated the local and systemic kinetics of TNF-α, IL-1ß, and IL-6 after injury in mice. We also hypothesized that injection of exogenous TNF-α, IL-1ß, and IL-6 would replicate the airway IgA response to injury. METHODS: : Experiment 1: male Institute of Cancer Research mice were randomized to uninjured controls (n = 8) or to surgical stress with laparotomy and neck incisions, with killing at 1, 2, 3, 5, or 8 hours after injury (n = 8/group). Bronchoalveolar lavage (BAL) and serum levels of TNF-α, IL-1ß, and IL-6 were analyzed by enzyme-linked immunosorbent assay. Experiment 2: male Institute of Cancer Research mice were randomized to uninjured controls (n = 6), injury (surgical stress that was similar to experiment 1 except the peritoneum was left intact, n = 6), or cytokine injection with intraperitoneal injection of recombinant TNF-α, IL-1ß, and IL-6. Animals were killed at 2 hours after injury, and nasal airway lavage and BAL IgA were analyzed by enzyme-linked immunosorbent assay. RESULTS: : Experiment 1: BAL TNF-α, IL-1ß, and IL-6 levels increased in bimodal pattern after injury at 3 hours and 8 hours versus controls (p < 0.05). Serum IL-6 did not increase at 3 hours, but did show a significant increase by 5 hours versus control (p < 0.05). Serum levels of TNF-α and IL-1ß did not change. Experiment 2: both Injury and combination TNF-α, IL-1ß, and IL-6 cytokine injection significantly increased IgA levels in airway lavage (BAL + nasal airway lavage) compared with control (p < 0.01 for both). CONCLUSIONS: : Airway levels of TNF-α, IL-1ß, and IL-6 increase in a bimodal pattern after injury with peaks at 3 hours and 8 hours, which do not correspond to serum changes. The peak at 8 hours is consistent with the known increase in airway IgA after injury. Intraperitoneal injection of a combination exogenous TNF-α, IL-1ß, and IL-6 replicates the airway IgA increase after injury. This effect is not seen with individual cytokine injections.